This proposal aims at enabling a device for direct low-input single molecule DNA and RNA sequencing. The device can be used to process picogram input levels, amounts that correspond to nucleic acid contents of individual cells. The readout technology is based on single molecule, real-time (SMRT) sequencing, where a copy of a DNA molecule is made by a polymerase enzyme, and optical detection of colors from four bases is employed to read the sequence in real time. In addition to DNA sequencing, detection of base modifications is possible by measuring the kinetics of base incorporation during replication. This technology has been documented by over 800 publications, and recently, a human genome assembly based entirely on SMRT sequencing data was reported. SMRT sequencing and nanopore-based sequencing are the two mainstream single-molecule sequencing technologies today, both offering long read lengths and direct DNA reads from native sample. However, neither technology is compatible with picogram-level input DNA, which is a serious limitation for analysis from single cells or small needle biopsies. Finally, direct RNA sequencing is of great interest for enabling direct transcriptome analysis without cDNA conversion and amplification, and for probing RNA base modifications, but there is no available method that can directly sequence RNA at the present time. In this collaboration between the Wanunu group at Northeastern University and Pacific Biosciences (PacBio), we combine our teams' unique expertise in enzyme engineering, organic synthesis, materials science, nanofabrication, surface chemistry, and single molecule optical detection, to solve the above challenges by allowing direct sequencing of DNA and RNA from picogram levels of input. The methods we will develop also allow for simultaneous detection of epigenetic base modification detection and resolve RNA secondary structures. Through a previous R21 grant received in 2012 the Wanunu group (in collaboration with PacBio) has developed nanopore-zero-mode waveguides, and further showed their use for efficient capture of picogram levels of DNA and RNA, and sequencing long DNA molecules. In this proposal, we describe a new type of device called a porous zero-mode waveguide (PZMW), which will address challenges in the previous device's design and scalability in order to reduce costs of fabrication by at least 2 orders of magnitude. In addition, based on promising preliminary results we will develop a method that will allow direct picogram-level RNA sequencing. In this method, enzyme engineering will be used to allow RNA template molecules to be replicated inside PZMWs, enabling the direct sequencing of full-length RNA transcripts with sensitivity to secondary structure and base modifications. We will demonstrate DNA and RNA sequencing from various low- input sources that come from our collaborators. Success in the proposed research will afford picogram-level DNA and RNA analysis with long read lengths, which would revolutionize genomics by enabling a deeper understanding of genomic, transcriptomic and epigenomic variation in disease and cell development.